Invasive Candida parapsilosis Bloodstream Infections in Children: The Antifungal Susceptibility, Clinical Characteristics and Impacts on Outcomes

Background: Candida parapsilosis is the most common non-albicans candida species that causes invasive candidiasis, but little is known about its impacts on the outcomes of pediatric patients. We aimed to characterize the clinical characteristics, risk factors and outcomes of C. parapsilosis bloodstream infections (BSIs) in children. Methods: All pediatric patients with Candida parapsilosis BSIs between 2005 and 2020 from a medical center in Taiwan were enrolled and analyzed. The antifungal susceptibility, clinical manifestations, management and outcomes were investigated. Cases of Candida parapsilosis BSIs were compared between patients with C. albicans BSIs and other Candida spp. BSIs. Results: During the study period, 95 episodes (26.0% of total cases) of Candida parapsilosis BSIs were identified and analyzed. No significant difference was found between pediatric patients with C. parapsilosis BSIs and those with C. albicans BSIs in terms of patients’ demographics, most chronic comorbidities or risk factors. Pediatric patients with C. parapsilosis BSIs were significantly more likely to have previous azole exposure and be on total parenteral nutrition than those with C. albicans BSIs (17.9 vs. 7.6% and 76.8 vs. 63.7%, p = 0.015 and 0.029, respectively). The duration of C. parapsilosis candidemia was relatively longer, and therefore patients often required a longer duration of antifungal treatment when compared with those of C. albicans candidemia, although the candidemia-attributable mortality rates were comparable. Of the C. parapsilosis isolates, 93.7% were susceptible to all antifungal agents, and delayed appropriate antifungal treatment was an independent factor in treatment failure. Conclusions: Pediatric patients with C. parapsilosis BSIs were more likely to have previous azole exposure and be on total parenteral nutrition, and the clinical significances included a longer duration of candidemia and patients often required a longer duration of antifungal treatment.


Introduction
Critically ill patients with long-term hospitalization in the intensive care unit (ICU), those with underlying immunocompromised status, the presence of artificial devices and the use of broad-spectrum antibiotics are vulnerable to Candida bloodstream infections (BSIs) [1][2][3]. A high mortality rate of 28-46% has been reported in pediatric patients with Candida BSIs, especially in extremely preterm neonates or those in the hematology/oncology

Study Design, Setting and Ethics Approval
All pediatric patients less than 18 years old who had Candida BSIs during hospitalization at the Linkou Chang Gung Memorial Hospital (CGMH) between January 2005 and December 2020 were enrolled and analyzed. Cases of Candida parapsilosis BSIs were compared with patients with C. albicans BSIs and other Candida spp. BSIs. The Linkou CGMH is the university-affiliated teaching hospital in northern Taiwan and the pediatric department of Linkou CGMH has several ICUs and a specialized hematology/oncology ward. There are a total of 24 beds and 107 beds in the pediatric ICU (PICU) and three neonatal intensive care units (NICUs) of Linkou CGMH, respectively. We continued the series of pediatric candidemia studies after the initial approval by the institutional Review Board of the CGMH (the certificate number: 202201214B0) over seven years ago. Additionally, a waiver of informed consent was also approved for anonymous data collection and the retrospective design of this study.

Definitions and Data Collection
In this study, we applied the following criteria to define Candida BSI: a patient had signs or symptoms of sepsis and ≥1 positive blood culture of Candida species. The Candida BSI episode was excluded if an unidentified Candida spp. was identified in the blood culture. In our institute, the clinicians usually repeat the blood cultures in cases of invasive candidiasis every 2 to 3 days until they are negative or when it is clinically indicated. The onset of Candida BSI was defined as the first positive blood culture of Candida spp. All bacterial pathogens or fungal species that were isolated while the patients were on antifungal therapy were reviewed and analyzed. If a bacterial pathogen was isolated within two days of the positive Candida spp. blood culture or two days after, it was considered as mixed Candida/bacteria BSIs. When a new infectious focus, such as an abscess, fungus ball, meningitis or end organ damage, were noted 48 h after the onset of the Candida BSI episode until the patient had completed the antifungal therapy or died, we considered it an infectious complication of the Candida BSI.
We applied the standard criteria of previous publications for the diagnosis of neonatal severe sepsis and septic shock [20,21]. When the patient completed antifungal therapy with at least two negative blood cultures from the last positive culture of the Candida isolate and resolution of all clinical symptoms, it was considered a new episode of Candida BSI if positive Candida spp. was isolated in the blood culture again [22]. Breakthrough candidemia was diagnosed if the new onset of Candida BSI occurred while this patient was still on antifungal therapy or antifungal prophylaxis [2,15].
Medical records were reviewed to determine response to antifungal therapy at two weeks after the onset of Candida BSIs, based on the guidelines for assessing treatment responses published by the Mycoses Study Group and the European Organization for Research and Treatment of Cancer as follows: complete response was the resolution of candidemia and clinical symptoms within 3 days; partial response was within 7 days; and progression of disease and death were considered "treatment failure" [23]. The demographic data, chronic comorbidities, hospital courses including use of antifungal agents and artificial devices and predisposing risk factors within 30 days before the onset of Candida BSI were also reviewed and analyzed. When the case had mortality before the resolution of signs and symptoms related to Candida BSIs or the patient died within 14 days after the onset of the Candida BSI without other explanation, it was defined as Candida BSI-attributable mortality [15,22]. For subsequent bacteremia following candidemia, it was defined as isolation of the bacterial pathogen between 48 h after the onset of Candida BSI and the time the patient completed antifungal therapy [23].

Microbiology and In Vitro Antifungal Susceptibility Testing
In Linkou CGMH, the BACTEC 9240 (Becton Dickinson Microbiology Systems, Franklin Lakes, NJ, USA) system is used to process all blood cultures. All Candida isolates from pediatric patients with Candida BSIs were retrieved from the central laboratory and re-identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF, Bruker Biotype, software version 3.0, Rochester, NY, USA). The in vitro antifungal susceptibilities of isolates were evaluated according to the EUCAST-Antifungal Susceptibility Testing microdilution method [24,25]. Candida krusei ATCC ® 6258 and Candida parapsilosis ATCC ® 22019 were used as quality control strains for antifungal drug susceptibility testing.

Statistical Analysis
All episodes of pediatric C. parapsilosis BSIs during the study period were analyzed and compared with all episodes of C. albicans BSIs in hospitalized children from our institute. The clinical characteristics, treatment and outcomes were compared between the two groups. The demographic, clinical, outcome variables and in vitro susceptibility data were summarized using descriptive statistics. Categorical variables were compared using the χ 2 or Fisher's exact test, and continuous variables by the Mann-Whitney U test. p-Values < 0.05 were considered statistically significant.
The clinical significance and impacts of pediatric Candida parapsilosis BSIs were investigated and independent risk factors for candidemia-attributable mortality were evaluated. A univariate logistic regression was fitted for each variable to test its relationship with mortality outcomes. Variables that were clinically relevant and statistically significant (p < 0.1) on univariate analysis were considered for the multivariate regression model. Clinical interventions were maintained in the final model as a fixed variable. All statistical analyses were performed using IBM SPSS software (version 22.0; IBM SPSS Inc., New York, NY, USA).

Results
In the study period, there were a total of 365 episodes of Candida BSIs in 320 pediatric patients hospitalized in our institute, with all the Candida isolates re-confirmed. There were 95 episodes of C. parapsilosis BSIs in 88 patients, accounting for 26.0% of all pediatric Candida BSIs in the study period. The most common Candida species that caused pediatric Candida BSIs were C. albicans (n = 171, 46.8%), followed by C. tropicalis (n = 21, 5.8%), C. glabrata (n = 20, 5.5%), and C. guilliermondii (n = 18, 4.9%). The trends of different Candida species that caused pediatric Candida BSIs during the study period are illustrated in Figure 1. The demographics and underlying chronic comorbidities of pediatric patients with C. parapsilosis BSIs are summarized in Table 1. Most of the pediatric patients (89.8%) had underlying chronic comorbidities at the onset of candidemia, and 44.3% had multiple chronic comorbidities. The distributions of C. parapsilosis BSIs between NICU, PICU or general wards and the infectious sources were comparable with those of C. albicans BSIs or other Candida species.
At the onset of candidemia, most C. parapsilosis BSIs were primary bloodstream infections, but 29.5% (n = 28) were catheter-related BSIs (CRBSI), and a total of 14 (14.7%) episodes had positive C. parapsilosis isolates cultured from the intra-abdominal space, abscess (n = 10), pleural fluid (n = 2) and urinary source (n = 2). The Candida isolates were identified from more than two sterile sites in four cases of C. parapsilosis BSIs and were considered as disseminated candidemia. (Table 2). A total of 17 episodes of C. parapsilosis BSIs were breakthrough candidemia; that is, the patients were on therapeutic antifungals or antifungal prophylaxis at their disease onset.  At the onset of candidemia, most C. parapsilosis BSIs were primary bloodstream infections, but 29.5% (n = 28) were catheter-related BSIs (CRBSI), and a total of 14 (14.7%) episodes had positive C. parapsilosis isolates cultured from the intra-abdominal space, abscess (n = 10), pleural fluid (n = 2) and urinary source (n = 2). The Candida isolates were identified from more than two sterile sites in four cases of C. parapsilosis BSIs and were considered as disseminated candidemia. (Table 2). A total of 17 episodes of C. parapsilosis BSIs were breakthrough candidemia; that is, the patients were on therapeutic antifungals or antifungal prophylaxis at their disease onset.

Microbiological Characteristics and Clinical Features
The antifungal susceptibility results of C. parapsilosis isolates are summarized in Table 3. The minimum inhibitory concentrations of C. parapsilosis (MIC 50 ) to fluconazole and voriconazole were 0.5 mg/L and 0.015 mg/L, respectively. All C. parapsilosis isolates were susceptible to fluconazole, voriconazole, amphotericin B and echinocandin-based antifungal regimens. There were only 1, 2 and 6 C. parapsilosis isolates that were resistant to itraconazole, micafungin and posaconazole, respectively. Overall, 93.7% C. parapsilosis isolates were susceptible to all antifungal agents.     Most of the clinical presentations, including severity of illness and percentages of severe sepsis, septic shock and disseminated candidemia, were comparable between C. parapsilosis BSIs and the control groups, including the C. albicans BSIs or other Candida spp. BSIs (Table 2). Most predisposing risk factors for candidemia were also comparable between the C. parapsilosis BSIs and the control groups. However, pediatric patients with C. parapsilosis BSIs were significantly more likely to have previous azole exposure and be on total parenteral nutrition (TPN) than those with C. albicans BSIs (17.9 vs. 7.6% and 76.8 vs. 63.7%, p = 0.015 and 0.029, respectively). Of note, the duration of C. parapsilosis BSIs was relatively longer than that of C. albicans BSIs Antifungal therapy was initiated after a median of 2 days (range, 0-7) after the onset of Candida parapsilosis BSIs. The initial antifungal agents and final therapeutic regimens were comparable between C. parapsilosis BSIs and C. albicans BSIs. The percentages of delayed appropriate antifungal agents and modifications of antifungal treatment were also comparable between the two groups. The median duration of antifungal treatments in patients with C. parapsilosis BSIs was 18.0 (IQR, 14-24) days, which was significantly longer than that of C. albicans BSIs (15.0 [14.0-22.0] days, p = 0.021). The longer duration of treatment was associated with a longer duration of candidemia and a higher rate of persistent candidemia in patients with C. parapsilosis BSIs. However, the candidemiaattributable mortality rates were comparable between C. parapsilosis BSIs, C. albicans BSIs and other Candida spp. BSIs. ( Table 4). The median time between the onset of the next nosocomial infection and the previous episode of C. parapsilosis BSIs was 10 days (range: 4-31 days).

Therapeutic Outcomes and Independent Risk Factors of Mortality
Overall, the attributable mortality rate of C. parapsilosis BSIs was 24.2% (23 of 95 episodes), and the in-hospital mortality rate was 33.0% (29 of 88 patients died). The therapeutic outcomes were not significantly different between different study periods, although echinocandins have been more commonly prescribed in our institute since 2010. There was no increasing trend or emergence of antifungal-resistant C. parapsilosis isolates during the study period in our cohort, although routine antifungal prophylaxis has been implemented for very low birth weight (VLBW, birth weight ≤ 1500 g) neonates in our NICUs since 2015. Additionally, the therapeutic responses were comparable between patients with C. parapsilosis BSIs and those with C. albicans BSIs and other Candida spp. BSIs.

Discussion
The trend of increasing non-albicans candidemia was noted in our institute and has also been documented in other countries in the literature [11,12,26]. Although C. parapsilosis BSIs in children were not significantly associated with worse outcomes, we found that pediatric patients with C. parapsilosis BSIs were more likely to have a longer duration of candidemia, slower responses to antifungal treatment and require a longer duration of antifungal treatment when compared with those with C. albicans BSIs. In children with Candida parapsilosis BSIs, the antifungal resistance rate was only 14% of all episodes [7], and only a few episodes had delayed initial appropriate treatment. We were unable to identify any independent risk factors for C. parapsilosis BSIs because most characteristics were comparable with the controls. However, C. parapsilosis candidemia deserves more concern since the high percentage of NICU patients with CVCs could potentially cause its increasing prevalence in the future.
The proportion of C. parapsilosis candidemia ranged between 14% and 34% of all Candida BSIs in both children and adults [11][12][13][14]18,26]. In recent years, non-albicans candidemia has outnumbered C. albicans candidemia, especially among critically ill patients, neonates with long-term CVC placement, those on TPN and those with antifungal prophylaxis [1,4,5,26,27]. Previous studies have found the use of CVC or other artificial devices, such as urinary catheters or mechanical ventilators, to be associated with C. parapsilosis candidemia [19,[28][29][30], while other studies also found patients receiving TPN were more likely to have C. parapsilosis CRBSI [11,31]. Our results were consistent with previous studies, although we could not find significant associations between CVC use and C. parapsilosis BSIs, which may be explained by the presence of CVC in almost all pediatric patients at the onset of candidemia. Additionally, a history of prior antifungal therapy was found to be associated with C. parapsilosis candidemia [30,32], which may be due to the effects of antifungal prophylaxis on the selection of non-albicans species, especially after echinocandin administration [32,33].
C. parapsilosis is known to exhibit a strong biofilm forming capability on the surfaces of a CVC or other artificial devices, which accounts for its higher prevalence among neonates with low birth weight, ICU patients with long term use of CVC and those who are immunocompromised [11][12][13][14]34]. Therefore, the most effective control strategy to avoid persistent candidemia is early catheter removal [7,16,34], although sometimes it is not applicable. Based on our results, we found C. parapsilosis BSIs had a significantly longer duration of candidemia, and delay in catheter removal was independently associated with final adverse outcomes. Previous studies have also found that patients with recurrent or breakthrough candidemia were more likely to have persistent candidemia and final treatment failure [15,35,36], which are supposed to be associated with the placement of CVCs. Given the inevitable requirement of a CVC in VLBW neonates, cases of C. parapsilosis BSIs deserve greater attention and further research for species-specific strategies is warranted.
Most studies found that patients with C. parapsilosis BSIs have comparable outcomes when compared with those with C. albicans BSIs or other non-parapsilosis candidemia [14,28,29,34], while others even found that the mortality rate was lower than that of C. albicans BSIs [37,38]. Overall, the different therapeutic strategies, study cohorts and different underlying demographics may account for the controversial results [11][12][13][14][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. The candidemia-attributable mortality rate in our cohort was relatively higher than that of previous studies [4,5,[30][31][32][33][34], especially in extremely preterm neonates. Although a new antifungal agent, echinocandin or caspofungin, has been launched in our institute since nearly a decade ago, this anti-biofilm agent did not significantly improve outcomes. We suspected that high percentages of chronic comorbidities, especially multiple chronic comorbidities, may account for the poor outcomes in our cohort. Although only a few C. parapsilosis isolates were antifungal-resistant, there has been an emergence of antifungal resistant C. parapsilosis isolates worldwide [39,40]. Therefore, C. parapsilosis BSIs deserves greater attention and specific therapeutic strategies for cases with a high risk of treatment failure should be investigated to optimize outcomes in the future.
There were some limitations in this study. The retrospective nature and single-center data limit the conclusion to being generalizable and applicable to other institutes or countries. Due to the long study period, there was inevitably some missing data and lost Candida parapsilosis isolates, which would make the incidence rate less reliable. The therapeutic strategies may have changed during the long study period, although the outcomes of C. parapsilosis BSIs were similar in the past two decades. Additionally, the pediatric cases of Candida BSIs in our institute did not have a regular schedule of follow-up blood cultures and the therapeutic strategies depended on the decisions of the attending physicians. Therefore, a further large-scale prospective study is required to address the risk factors and real impacts of C. parapsilosis on the outcomes of pediatric patients with Candida BSIs.
In conclusion, C. parapsilosis isolates are the most common non-albicans candidemia in children, and the use of CVC and other artificial devices, as well as the current antifungal prophylaxis policy, may predispose children to have C. parapsilosis BSIs. The mortality rate of pediatric patients with Candida BSIs in children was high, and the chance of increasing azole resistance in Candida isolates deserves more concern. Since early catheter removal is especially important to avoid persistent candidemia and significantly affect final outcomes, clinicians should not treat C. parapsilosis BSIs with catheters in situ. In cases of multiple chronic comorbidities or septic shock, clinicians should consider more aggressive therapies.  Institutional Review Board Statement: This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital with a waiver of informed consent because all patient records and information were anonymized and deidentified prior to analysis.
Data Availability Statement: The datasets used/or analyzed during the current study are available from the corresponding author on reasonable request.